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. 2022 Aug 29;41(20):e111161. doi: 10.15252/embj.2022111161

Figure 4. SLC15A4 promotes inflammasome perinuclear positioning away from autophagic membranes.

Figure 4

  • A–G
    WT or SLC15A4 feeble BMDCs expressing ASC‐GFP and mCherry‐LC3 (A, B, D) or non‐transduced (C) or SLC15A4 feeble BMDCs transduced with SLC15A4‐GFP (E, F, G) were infected with flagellin‐expressing STm and fixed (A–F) or lysed (G) 1 h after infection. (A) Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry‐LC3 (red) in two infected WT and SLC15A4 feeble DCs each. (B) Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of three independent experiments. (C) Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4 feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4 feeble DCs. (D) Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of three independent experiments. (E, F) Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4‐GFP (green) in SLC15A4 feeble DCs together with non‐transduced SLC15A4 feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non‐perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). (G) Representative immunoblots showing pro‐caspase‐1 (pro‐casp.‐1) and cleaved p20 (casp.‐1 p20), LC3‐II and LC3‐I, and actin bands. N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.

Data information: Data represent mean ± SD. **P < 0.01. Two‐tailed Student's t‐test. See also Fig EV4.

Source data are available online for this figure.